The present invention relates generally to polymers having controlled architectures, to controlled free-radical polymerization methods for preparing such polymers, to separation media prepared from such polymers, and to separation applications for such media. The invention relates, more specifically, to non-linear polyacrylamidic polymers having useful properties, such as controlled weight-average molecular weights, narrow polydispersity indices, flow-enabling viscosities, and water- or aqueous medium-solubility or dispersability, and to flowable separation media prepared from such polymers for use in capillary electrophoresis. The invention also specifically relates to living-type, nitroxide-mediated free-radical polymerization for preparing such polymers, as well as other polymers, in aqueous solution.
Electrophoresis and/or electroosmotic flow is a technique used for separation and analysis of charged molecules such as biopolymers (e.g., nucleic acid polymers such as DNA, RNA and amino acid polymers such as proteins). Typically, one or more samples containing molecules to be separated or analyzed are loaded onto a separation media, and a voltage is applied across the media. The applied voltage causes the charged molecules to move differentially, thereby fractionating the sample into its various components.
Gel electrophoresis typically employs a stationary, relatively flat separation media, referred to in the art as a xe2x80x9cslab-gelxe2x80x9d, and usually comprising a highly cross-linked polymer. Gel electrophoresis enhances separation of macromolecules by introducing a sieving effect, which helps separate such molecules according to size, and which complements separation according to electrophoretic mobilities. In capillary gel electrophoresis, a voltage is generally applied across a capillary that comprises a separation media. The separation medium can be stationary or flowing relative to the capillary. Typically, stationary separation media comprises either a crosslinked gel, a polymer solution, or other flowable media. Samples such as biomolecules are loaded or otherwise disposed onto or into stationary or flowing separation media. The applied voltage causes the biomolecules to differentially migrate, relative to the separation media, to a detector. The rate at which the biomolecule migrates depends upon a number of factors including the nature of the biomolecule, the size and weight of the biomolecule, the charge on the biomolecule, the nature and properties of the separation media, and the conditions under which the separation is performed.
Capillary electrophoretic separation approaches have been automated, but such approaches have limitations with respect to sample component resolution. Known separation media for capillary gel electrophoresis typically comprise linear molecular chains that may become entangled and thereby impart a mesh-like characteristic to the separation media. The pore-size of such an entangled mesh is dynamic such that the size and persistence time of the mesh in a linear polymer is related to both the chain length and the number of chains per unit volume. Hence, both polymer molecular weight and polymer concentration will impact the separation capabilities of the media for different sample components (e.g., different lengths of DNA). Resolution of relatively smaller sample molecules is more effective with a more closely-knit meshxe2x80x94requiring lower molecular weight chains and/or higher polymer concentrations, whereas higher molecular weights are preferred for larger molecules. However, known separation media for capillary electrophoresis become unsuitably viscous when higher-molecular weight polymers and/or higher concentrations of polymer are employed in such media, sometimes referred to as replaceable media.
Overly-viscous media are generally disadvantageous in capillary electrophoresis systems due to the reduced flowability of the media, and due to the increased shear stresses between polymer molecules, and between polymer molecules and the capillary wall. In particular, shear stresses can interfere with the electrophoretic analysis and can cause polymer degradation. In addition, high viscosity may result in microbubble-spikes in the electropherogram. The relatively high viscosity of known separation media also presents a substantial barrier to improving performance and versatility in smaller scale capillary gel electrophoresis systemsxe2x80x94particularly, for example, for micro-scale applications thereof, known in the art as microelectrophoresis systems.
The separation media for capillary electrophoresis have also been limited, heretofore, by the lack of narrow-band polymers for use in such media. Polymers typically comprise a number of different polymer molecules, each of which comprises substantially the same type of repeat unit(s) but which can vary in the number of repeat units (i.e., chain length). The polydispersity indexxe2x80x94the ratio of the weight-average molecular weight to the number-average molecular weight for the polymer (Mw/Mn), is a measure of the homogeneity of the polymer with respect to hydrodynamic volume, and therefore, for linear polymers, with respect to chain-length. The state-of-the-art separation media for capillary gel electrophoresis comprise polymers having a polydispersity index greater than two, and typically ranging from about 3 to about 4. Moreover, the use of polymers with relatively high polydispersity indices limits the degree of flexibility available for formulating separation mediaxe2x80x94particularly for applications such as capillary gel electrophoresis where viscosity constraints apply.
Although present commercial capillary gel electrophoresis separation media comprise linear polymers, non-linear, star-shaped block copolymers have also been contemplated for use in such separation media. Specifically, U.S. Pat. No. 5,290,418, U.S. Pat. No. 5,759,369 and U.S. Pat. No. 5,468,365 to Menchen et al. report lower molecular weight star-copolymers having arms comprising alternating, regularly repeating, hydrophobic segments (e.g., fluorinated hydrocarbons) and hydrophilic segments (e.g., polyethylene oxide). A sieving effect is apparently achieved based on association between the hydrophobic regions of polymer segmentsxe2x80x94forming a physical, non-covalent cross-link. The attachment polymerization methods employed by Menchen et al., however, lead to polymers that are polydisperse, are limited with respect to yields for attaching long polymer chains onto a common central core (e.g., are of low molecular weight), and are constrained with respect to the choice of monomer.
Hence, there remains a need in the art for improved polymeric materials suitable for use in connection with capillary gel electrophoresis.
Living-type free-radical polymerization is generally known in the art. Mediation of such free-radical polymerization using nitroxide free-radical control agents is also known for some applications.
It is, therefore, an object of the invention to provide separation media having improved separation capabilities. It is also an object of the invention to provide controlled-architecture polymers having useful properties. It is likewise an object of the invention to demonstrate controlled free-radical polymerization in aqueous solutions and in bulk.
In particular, it is a feature of this invention that low viscosity polymers, yet of high molecular weight, are provided. Due to the surprising relationship between the molecular weight of the polymer in the separation media and the ability of the separation media to separate increasing numbers of base pairs, high molecular weight polymers are particularly desirable. This invention thus meets the particular requirements for separation media for biological molecule separation. This invention preferably provides non-linear, controlled architecture polymers that have high molecular weights, but sufficiently low viscosity such that the polymers can be incorporated into separation media in higher concentrations than previously practical.
Briefly, therefore, the present invention is directed to polymers, the preparation of polymers, blends of polymers, separation media, the preparation of separation media, capillary gel electrophoresis apparatus comprising such media, and analysis of samples by capillary gel electrophoresis, as described and set forth herein.
The controlled-architecture polymers of the present invention can be employed to prepare improved separation media for capillary gel electrophoresis. In particular, these polymers provide a substantial degree of flexibility for separation media formulationxe2x80x94particularly with respect to molecular weight, viscosity, polydispersity index and polymer loading (i.e., weight concentration), among other important properties. As such, it is now possible to prepare separation media suitable for separation and/or analyses of samples having broadly varying component characteristics. It is likewise possible to prepare tailored separation media for more specific separations and/or analyses of particular samples of interest. In addition, performance characteristics, such as sample throughput, resolution and peak capacity can be significantly enhanced. Moreover, the substantially reduced viscosity of the controlled architecture polymers of the invention offer substantial advantages for applications such as separation media for capillary gel electrophoresisxe2x80x94particularly for micro-scale applications thereof (i.e., microelectrophoresis).
Other features, objects and advantages of the present invention will be in part apparent to those skilled in art and in part pointed out hereinafter. All references cited in the instant specification are incorporated by reference for all purposes. Moreover, as the patent and non-patent literature relating to the subject matter disclosed and/or claimed herein is substantial, many relevant references are available to a skilled artisan that will provide further instruction with respect to such subject matter.